Single-Molecule Localization Microscopy of 3D Orientation and Anisotropic Wobble using a Polarized Vortex Point Spread Function

Within condensed matter, single fluorophores are sensitive probes of their chemical environments, but it is difficult to use their limited photon budget to image precisely their positions, 3D orientations, and rotational diffusion simultaneously. We demonstrate the polarized vortex point spread function (PSF) for measuring these parameters, including characterizing the anisotropy of a molecule’s wobble, simultaneously from a single image. Even when imaging dim emitters (∼500 photons detected), the polarized vortex PSF is able to obtain 12 nm localization precision, 4-8° orientation precision, and 26° wobble precision. We use the vortex PSF to measure the emission anisotropy of fluorescent beads, the wobble dynamics of Nile red (NR) within supported lipid bilayers, and the distinct orientation signatures of NR in contact with amyloid-beta fibrils, oligomers, and tangles. The unparalleled sensitivity of the vortex PSF transforms single-molecule microscopes into nanoscale orientation imaging spectrometers, where the orientations and wobbles of individual probes reveal structures and organization of soft matter that are nearly impossible to perceive using molecular positions alone.

Поляризованная люминесценция наноточек MoS-=SUB=-2-=/SUB=-

The effect of temperature on the polarization of the luminescence of the colloidal system of MoS2 nanodots in n-methylpyrrolidone is studied under the condition of linearly polarized excitation. Nanodots are obtained by chemical exfoliation and dispersion of MoS2 microcrystals in a liquid medium under the action of ultrasound. The photoluminescence spectrum of the ensemble of MoS2 nanodots is significantly shifted towards shorter wavelengths with respect to the luminescence spectrum of bulk crystals, which is explained by the quantum-size effect in the electronic spectrum of MoS2 nanocrystals. It is shown that the temperature dependence of the anisotropy of the radiation of nanodots is described by the Levshin-Perrin equation, which takes into account the rotational diffusion of luminescent particles in the liquid matrix. The size of photoexcited nanodots in the framework of the Levshin-Perrin model turns out to be ≥ 1.5 nm and increases with increasing the emission wavelength. It is shown that the sizes of MoS2 nanodots obtained by analyzing the temperature dependence of the emission anisotropy are in satisfactory agreement with the data obtained by analyzing the quantum-size effect in the electronic spectrum of nanodots.

Polarization-resolved single-molecule tracking reveals strange dynamics of individual fluorescent tracers through a plasticized (rubbery) polymer network

<div>Tracking the movement of fluorescent single-molecule (SM) tracers has provided several new insights on the local structure and dynamics in complex environments such as soft materials and biological systems. However, SM tracking (SMT) remains unreliable at molecular length scales, as the localization-error (LE) of SM trajectories (~30-50 nm) is considerably larger than size of molecular tracers (~1-3 nm). Thus, instances of tracer (im)mobility in heterogeneous media, which provide indicator for underlying anomalous-transport mechanisms, remains obscured within the realms of SMT. Since translation of passive tracers in an isotropic network is associated with fast dipolar rotation, we propose authentic pauses within LE can be revealed upon probing SM reorientational dynamics. Here, we demonstrate how polarization-resolved SMT (PR-SMT) can provide emission-anisotropy at each super-localized position, thereby revealing tumbling propensity of SMs during random walk. For Rhodamine 6G tracers undergoing heterogeneous transport in a hydrated polyvinylpyrrolidone (PVP) network, analyses of PR-SMT trajectories enabled us to discern instances of genuine immobility and localized motion within the LE. Our investigations on 100 SMs in hydrated (plasticized) PVP films reveal a wide distribution of dwell-times and pause-frequencies, which demonstrate that majority of probes intermittently experience complete translational and rotational immobilization. This indicates tracers serendipitously encounter compact, rigid polymer cavities during transport, implying the existence of nanoscale glass-like domains sparsely distributed in a redominantly deep-rubbery polymer network far above the glass transition. PR-SMT is simple to implement and opens up alternate avenues to interrogate transient (bio)molecular interactions leading to anomalous transport in inhomogeneous media.</div>

Polarization-resolved single-molecule tracking reveals strange dynamics of individual fluorescent tracers through a plasticized (rubbery) polymer network

<div>Tracking the movement of fluorescent single-molecule (SM) tracers has provided several new insights on the local structure and dynamics in complex environments such as soft materials and biological systems. However, SM tracking (SMT) remains unreliable at molecular length scales, as the localization-error (LE) of SM trajectories (~30-50 nm) is considerably larger than size of molecular tracers (~1-3 nm). Thus, instances of tracer (im)mobility in heterogeneous media, which provide indicator for underlying anomalous-transport mechanisms, remains obscured within the realms of SMT. Since translation of passive tracers in an isotropic network is associated with fast dipolar rotation, we propose authentic pauses within LE can be revealed upon probing SM reorientational dynamics. Here, we demonstrate how polarization-resolved SMT (PR-SMT) can provide emission-anisotropy at each super-localized position, thereby revealing tumbling propensity of SMs during random walk. For Rhodamine 6G tracers undergoing heterogeneous transport in a hydrated polyvinylpyrrolidone (PVP) network, analyses of PR-SMT trajectories enabled us to discern instances of genuine immobility and localized motion within the LE. Our investigations on 100 SMs in hydrated (plasticized) PVP films reveal a wide distribution of dwell-times and pause-frequencies, which demonstrate that majority of probes intermittently experience complete translational and rotational immobilization. This indicates tracers serendipitously encounter compact, rigid polymer cavities during transport, implying the existence of nanoscale glass-like domains sparsely distributed in a redominantly deep-rubbery polymer network far above the glass transition. PR-SMT is simple to implement and opens up alternate avenues to interrogate transient (bio)molecular interactions leading to anomalous transport in inhomogeneous media.</div>

In-Parallel Polar Monitoring of Chemiluminescence Emission Anisotropy at the Solid–Liquid Interface by an Optical Fiber Radial Array

Chemiluminescence (CL) detection is widely employed in biosensors and miniaturized analytical devices since it offers high detectability and flexible device design (there are no geometry requirements for the measurement cell, except the ability to collect the largest fraction of emitted photons). Although the emission anisotropy phenomenon for an emitting dipole bound to the interface between two media with different refractive index is well known for fluorescence, it is still poorly investigated for CL reactions, in which the excited-state reaction products can diffuse in solution before the photon emission event. In this paper, we propose a simple method for the real-time evaluation of the CL emission anisotropy based on a radial array of optical fibers, embedded in a poly(methyl methacrylate) semicylinder and coupled with a Charge-Coupled Device (CCD) camera through a suitable interface. The polar-time evolutions of the CL emission have been studied for catalyzing enzymes immobilized onto a solid surface (heterogeneous configuration) or free in solution (homogeneous configuration). Evidence of the anisotropy phenomenon is observed, indicating that the lifetime of the excited-state products of the enzyme-catalyzed reactions is shorter than the time required for their diffusion in solution at a distance at which the CL can be considered isotropic. These results open new perspectives in the development of CL-based miniaturized analytical devices.

Cosmic ray interactions in the solar atmosphere

ABSTRACT High-energy particles enter the solar atmosphere from Galactic or solar coronal sources, and produce ‘albedo’ emission from the quiet Sun that is now observable across a wide range of photon energies. The interaction of high-energy particles in a stellar atmosphere depends essentially upon the joint variation of the magnetic field and plasma density, which heretofore has been characterized parametrically as P ∝ Bα with P the gas pressure and B the magnitude of the magnetic field. We re-examine that parametrization by using a self-consistent 3D MHD model (Bifrost) and show that this relationship tends to P ∝ B3.5 ± 0.1 based on the visible portions of the sample of open-field flux tubes in such a model, but with large variations from point to point. This scatter corresponds to the strong meandering of the open-field flux tubes in the lower atmosphere, which will have a strong effect on the prediction of the emission anisotropy (limb brightening). The simulations show that much of the open flux in coronal holes originates in weak-field regions within the granular pattern of the convective motions seen in the simulations.

DISTINCT ACTIN-DEPENDENT NANOSCALE ASSEMBLIES UNDERLIE THE DYNAMIC AND HIERARCHICAL ORGANIZATION OF E-CADHERIN

SUMMARYIntercellular adhesion mediated by E-cadherin is pivotal in maintaining epithelial tissue integrity and for tissue morphogenesis. Adhesion requires homophilic interactions between extracellular domains of E-cadherin molecules from neighboring cells. The interaction of its cytoplasmic domains with the cortical acto-myosin network, appears to strengthen adhesion, although, it is unclear how cortical actin affects the organization and function of E-cadherin dynamically. Here we use the ectopic expression of Drosophila E-cadherin (E-cad) in larval hemocytes, which lack endogenous E-cad, to recapitulate functional cell-cell junctions in a convenient model system. We used fluorescence emission anisotropy-based microscopy and Fluorescence Correlation Spectroscopy (FCS) to probe the nanoscale organization of E-cad. We find that E-cad at cell-cell junctions in hemocytes exhibits a clustered trans-paired organization, similar to that reported for the adherens junction in the developing embryonic epithelial tissue. Further, we find that extra-junctional E-cad is also organized as relatively immobile nanoclusters as well as diffusive and more loosely packed oligomers and monomers. These oligomers are promoted by cis-interactions of the ectodomain and, strikingly, their growth is constantly counteracted by cortical actomyosin. Oligomers in turn assist in generating nanoclusters that are stabilized by cortical acto-myosin. Thus, actin remodels oligomers and stabilizes nanoclusters, revealing a requirement for actin in the dynamic organization of E-cad at the nanoscale. This dynamic organization is also present at cell-cell contacts (junction), and its disruption affects junctional integrity in the hemocyte system, as well as in the embryo. Our observations uncover a hierarchical mechanism for the nanoscale organization of E-cad, which is necessary for dynamic adhesion and maintaining junctional integrity in the face of extensive remodeling.